Method of treating skin diseases

ABSTRACT

A group of hydro-monobenzoporphyrins &#34;green porphyrins&#34; (Gp) having absorption maxima in the range of 670-780 nanometers is useful in treating disorders or conditions which are subject to hematoporphyrin derivative (HPD) treatment in the presence of light, or in treating virus, cells and tissues generally to destroy unwanted targets. The use of the Gp of the invention permits the irradiation for therapy to use wavelengths other than those absorbed by blood. The Gp of the invention may also be conjugated to ligands specific for receptor or to specific immunoglobulins or fragments thereof to target specific tissues or cells for the radiation treatment. Use of these materials permits lower levels of drug to be used, thus preventing side reactions which might destroy normal tissues.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application is a division of application Ser. No. 07/943,895 filedSep. 11, 1992, now U.S. Pat. No. 5,283,255, which is a division of Ser.No. 07/718,393 filed Jun. 20, 1991, now U.S. Pat. No. 5,171,749, whichis a continuation-in-part of U.S. Ser. No. 414,201, filed Sep. 28, 1989,now U.S. Pat. No. 5,095,030, which is a continuation-in-part of U.S.Ser. No. 221,161, filed Jul. 19, 1988, now U.S. Pat. No. 4,920,143,which is a continuation-in-part of U.S. Ser. No. 041,680, filed Apr. 23,1987, now U.S. Pat. No. 4,883,790, which is a continuation-in-part ofU.S. Ser. No. 005,204, filed Jan. 20, 1987, now abandoned.

FIELD OF THE INVENTION

The invention relates to the use of light absorbing compounds to mediatethe destruction of unwanted cells or tissues or other undesirablematerials by irradiation or to detect their presence throughfluorescence. Specifically, the invention relates to the use ofhydro-monobenzoporphyrin derivatives having absorption maxima in therange 670-780 nanometers to mediate the irradiation of materials to bedestroyed, and to the use of these compounds conjugated totarget-specific ligands, such receptor-specific ligands, orimmunoglobulins or their immunospecific fragments, to focus the effectsof the irradiation on particular targets.

BACKGROUND OF THE INVENTION

The use of hematoporphyrin and its acetylated derivative mixturehematoporphyrin derivative (HPD) systemically, combined withirradiation, for the detection and treatment of malignant cells has, bythis time, some considerable history. HPD is a mixture of porphyrinsincluding hematoporphyrin itself, hydroxyethyl vinyl deuteroporphyrin,protoporphyrin, and dihematoporphyrin ethers. (See, e.g., "PorphyrinPhotosensitization," Kessel, D., et al., eds. (1983) Plenum Press.)

HPD seems "naturally" capable of localizing in malignant cells. Whenirradiated, it has two properties which make it useful. First, whenirradiated with ultraviolet or visible light, it is capable offluorescence, and thus is useful in diagnostic methods related todetection of malignancy (see, for example, Kessel, D., et al. (supra);Gregory, H. B., Jr., et al., Ann Surg (1968) 167:827-829). Alsoimportant is the capacity of HPD, when irradiated with visible light, toexhibit a cytotoxic effect on the cells or other tissue in which it islocalized (see, for example, Diamond, I., et al., Lancet (1972)2:1175-1177; Dougherty, T. J., et al., Cancer Research (1978)38:2628-2635; Dougherty, T. J., et al., "The Science of Photo Medicine"(1982) J. D. Regan & J. A. Parrish, eds., pp. 625-638; Dougherty, T. J.,et al., "Cancer: Principles and Practice of Oncology" (1982) V. T.DeVita Jr., et al., eds., pp. 1836-1844). Although it has not beendefinitively established, the effect of HPD in killing cells seems to bedue to the formation of singlet oxygen upon irradiation (Weishaupt, K.R., et al., Cancer Research (1976) 36:2326-2329). Several mechanisms forthis effect have been proposed, and it has been shown that the activeingredient(s) in HPD which mediates the cytotoxic effect of visiblelight irradiation is the mixture of dihematoporphyrin ethers (DHE)(Dougherty, T. J., et al., "Porphyrin Localization and Treatment ofTumors" (1984) pp. 301-314; Dougherty, T. J., CRC Critical Reviews inOncology/Hematology (1984) 2:83-116).

A purified form of the active component(s) of HPD is obtained byadjustment of pH to cause aggregation and recovery of the aggregate, asdisclosed in U.S. Pat. Nos. 4,649,151, 4,866,168, 4,889,129 and4,932,934. The purified form called DHE in the patent, is marketed underthe trademark Photofrin® II and has been used in a manner completelyanalogous to HPD.

In addition to in vivo therapeutic and diagnostic protocols for tumorsas described in the above-cited patent, the porphyrins, including HPDand its more purified derivatives, can be used in other in vivo and invitro applications. For example, photosensitizers are useful in thedetection and treatment of atherosclerotic plaques as described in U.S.Pat. Nos. 4,512,762 and 4,577,636. U.S. Pat. No. 4,500,507 and 4,485,806describe the use of radiolabeled porphyrin compounds, including HPD, fortumor imaging. U.S. Pat. No. 4,753,958 to the University of Californiadescribes the use of topical application of porphyrin sensitizers fordiagnosis and treatment of skin diseases. U.S. Pat. Nos. 4,748,120 and4,878,891 describe the use of photosensitizers in the treatment of wholeblood or blood components. Photochemical decontamination treatment ofblood and components is also described in U.S. Pat. No. 4,727,027 wherethe photosensitizer is furocoumarin and its derivatives. In addition,viruses are inactivated in therapeutic protein compositions in vitro asdisclosed in U.S. Pat. No. 4,268,947.

The successful treatment of AIDS-related oral Kaposi's Sarcoma with therelated photosensitizer Photofrin® II porfimer sodium was described bySchweitzer, V. G., at al., Otolaryngology--Head and Neck Surgery (1990)102:639-649. It is expected that the modified porphyrins of the presentinvention will also be effective in regard to this indication and thatthe sole related side effect--hypersensitivity to sunlight--will beavoided due to the lower dose levels required for the inventioncompounds.

While the treatment of tumors and other undesirable targets with HPDrelies on the intrinsic ability of HPD to localize in malignant or otherrelevant cells or targets, a considerable improvement and refinement inspecificity has been achieved by conjugating the hematoporphyrin totarget-specific antibodies. For example, when hematoporphyrin wascoupled to monoclonal antibodies directed to a murine myosarcoma cellline M1, administration of anti-M1 hematoporphyrin-conjugates totumor-bearing animals followed by exposure to incandescent lightresulted in the suppression of M1 growth (Mew, D., et al., J Immunol(1983) 130:1473-1477). In additional work, hematoporphyrin wasconjugated to a monoclonal antibody specific to an antigen associatedwith a human leukemia (CAMAL) and the conjugates were shown to mediatethe irradiation-induced killing of leukemic cells specifically, in vitro(Mew, D., et al., Cancer Research (1985) 45:4380-4386). Conjugation ofthe related compound chlorin e₆ to anti-T cell Mab has also beenreported (Oseroff, A. R., et al., Proc Natl Acad Sci U.S.A. (1986)83:8744-8748).

While the conjugation of hematoporphyrin to immunoglobulins specific fortargeted cells refines the ability of the hematoporphyrin to home to thedesired cells or tissue, this still does not solve another problemancillary to this general therapeutic approach, namely that thewavelength for irradiation required to activate the hematoporphyrin orHPD, which is in the range of 630 nanometers, is also an energy which isreadily absorbed by the endogenous porphyrins and other naturalchromophores in the blood and other tissues. Therefore, relatively largeamounts of the hematoporphyrin or HPD must be administered, oftenresulting in oversensitization of the patient to light in general. Itwould be desirable to administer compounds to mediate the effects ofirradiation in a lower amount and with higher clearance rates, thusavoiding the problems of hypersensitivity exhibited nonspecificallythroughout the subject organism. The activity of certain of thesecompounds was described in a paper by Richter, A. M., et al., in J NatlCancer Inst (1987) 79:1327-1332, mailed to subscribers on Jan. 19, 1988.The invention is directed to the use of such compounds.

DISCLOSURE OF THE INVENTION

The invention provides light absorbing compounds capable of exhibitinglight-mediated cytotoxic and diagnostic effects. In addition to their invitro use, these compounds may be administered in in vivo relatively lowdosage due to their capability to absorb radiation whose energy range isoutside of that normally strongly absorbed by the components present inhigh concentration in the blood or other tissues, in particular, theporphyrin residues normally associated with hemoglobin and myoglobin.Therefore, by providing these modified porphyrins for in vivo treatmentat lower concentration, hypersensitivity of nontarget tissues isreduced, and the irradiation treatment can be conducted at a wavelengthat which the native chromophores do not compete as effectively forphotons with the active compounds, resulting in greater depth ofpenetration of the light. Similar advantages accrue in in vitrotreatment of colored materials, such as blood samples.

These photoactive compounds are modified porphyrins which, by virtue oftheir derivatization, undergo a shift in absorption maxima so that theyappear green rather than red, indicating their absorption of wavelengthsin the red-orange range. This collection of derivatives has thereforebeen nicknamed "green porphyrin" (Gp) and has been shown to confersensitivity on target cells at concentrations greater than 10-fold lowerthan those required for hematoporphyrin (Hp) or HPD.

The Gp is selected from a group of porphyrin derivatives obtained usingDiels-Alder reactions of acetylene derivatives with protoporphyrin underconditions which effect a reaction at only one of the two availableconjugated, nonaromatic diene structures present in theprotoporphyrin-IX ring system (rings A and B). The formulas shown inFIG. 1 represent the green porphyrins of the invention. These compoundsare shown in the figure with hydrogen occupying the internal ringnitrogens; however, it is understood that the metalated forms wherein acation replaces one or both of these hydrogens can also be employed. Itis also understood that these compounds can be labeled either byreplacement of one or more of the atoms in the structure by itsradioactive form, or by coupling to a radioisotope such as a radioactivemetal or, for example, a radioisotope of iodine.

For convenience, an abbreviation of the term hydro-monobenzoporphyrinderivative--"BPD"--is generally used to refer to compounds of formulas 3and 4 of FIG. 1, as these are the preferred forms of Gp.

Furthermore, dimeric forms of the Gp can be provided, thus amplifyingthe ability of the Gp compound to absorb light on a per mole basis.Dimeric and multimeric forms of Gp/porphyrin combinations can also beemployed, providing additional absorption wavelengths.

The modified porphyrins (referred to as "green porphyrin" or "Gp"herein) of the invention can be conjugated to specific ligands reactivewith a target, such as receptor-specific ligands or immunoglobulins orimmunospecific portions of immunoglobulins, permitting them to be moreconcentrated in a desired target tissue or substances. This conjugationpermits further lowering of the required dose levels since the materialis not wasted in distribution into other tissues whose destruction, farfrom being desired, must be avoided.

Thus, in one aspect, the invention relates to methods of locating targetmethods or effecting cytotoxicity by photosensitizing the targetmaterials using the hydro-monobenzoporphyrins of the invention eitheralone or as conjugates. The hydro-monobenzoporphyrins are greenporphyrins (Gp) as shown in FIG. 1 or their metalated or labeled forms,and are localized specifically in vivo to certain target tissues, wheretheir presence can be detected by fluorescence upon excitation usingabsorbed wavelengths, or by other means when the Gp is provided withadditional or alternate labeling. As indicated above, the specificity ofthe Gp can be further enhanced by conjugation to ligands specific forthe target. In addition, when the Gp is irradiated in situ using lightin the visible absorption range, photoactivation results in cytotoxicityto the surrounding tissue. While the absorption spectrum also includesshorter wavelengths, there is an especially useful absorption maximum inthe 670-780 nm range. Cells to which the Gp is normally attractedinclude tumor cells, and neoplastic cells in general, as well asbacteria, virus, atherosclerotic plaque, restenotic tissue, lesions andother diseased tissues. The method can be applied either in vivo or invivo, and, when applied in vivo, can be localized, including topical, orsystemic, including oral, administration.

In another aspect, the invention relates to certain specific Gpcompounds including those of formulas 3 and 4 designated herein "BPD,"that are partially hydrolyzed forms containing free (non-esterified)carboxylic acid moieties or their salts in the R³ substituents. Theinvention also relates to labeled forms of these compounds.

In other aspects, the invention relates to conjugates of the formulasRe*-L-Gp and Ig-L-Gp wherein Re* represents a ligand which is specificto, and capable of, binding a receptor at a cell surface, Ig representsan immunoglobulin or an immunologically reactive portion thereof, Gprepresents a hydro-monobenzoporphyrin having an absorption maximum inthe range of 670-780 nanometers, and L represents either a covalent bondlinking these components or a linking moiety covalently linked to eachof the Re* or Ig and Gp.

The invention is also directed to tripartite complexes which includeRe*-L-Gp or Ig-L-Gp further conjugated to or associated with a label.The label may be bound either to the targeting component or to the Gp orboth.

In another aspect, the invention relates to pharmaceutical compositionscontaining these active ingredients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-1 and 1-2 show the structure of green porphyrin (Gp) compoundsused in the methods and conjugates of the invention.

FIGS. 2-1 and 2-2 show the structure of four preferred forms of thehydro-monobenzoporphyrin derivative of formulas 3 and 4 (BPDs).

FIG. 3 shows a comparative absorption spectrum of a BPD compound andprior art compositions.

FIG. 4 shows the results of skin sensitivity assay using a BPD compound.

FIGS. 5, 6 and 7 graphically represent the effect of BPD concentrationand light exposure times on viral kill in plasma (FIG. 5) and red cellconcentrations (FIGS. 5, 6 and 7) in the LED activation study of Example19.

FIGS. 8, 9 and 10 also derive from the LED activation study of Example19, and demonstrate results achieved with an increase in lightintensity.

MODES OF CARRYING OUT THE INVENTION The Hydro-monobenzoporphyrins (Gp)

All of the compositions of the invention employ as the light absorbingcompound, a derivative of the protoporphyrin ring system which has alight absorption maximum in the range of 670-780 nanometers. FIG. 3shows the absorption spectrum of one of the compounds of the inventionshown in FIG. 2, BPD-DA, wherein R¹ and R² are carbomethoxy, incomparison to HPD and Photofrin® II compositions. Only BPD-DA has amajor absorption peak at about 685 nm.

In general, this shift is achieved by effectively saturating one of thetwo π-bonds in one, but not two, of the four pyrrole rings whichconstitute the typical porphyrin system. In protoporphyrin-IX two of thepyrroles contain vinyl substitutions such that the exocyclic π-bond isconjugated to one of the two π-bonds in the ring. A Diels-Alder reactioninvolving one of these conjugated systems with an acetylene derivativedienophile results in a fused cyclohexadiene--referred to herein as"hydrobenzo"--fused to the A or B ring, as shown in formulas 1 and 2.Rearrangement of the π system in the hexadiene ring results in thecompounds of formulas 3 and 4; reduction provides the compounds offormulas 5 and 6. All of these compounds provide the desiredbathochromic shift in absorption maximum.

Specific preparation of some compounds useful in the invention or theirprecursors is described by Morgan, A. R., et al., J Chem Soc Chem Commun(1984) pp. 1047-1048; and by Pangka, B. S., et al., J Organic Chem(1986) 51:1094. As described in these publications, it had earlier beenreported that protoporphyrin-IX dimethyl ester, when reacted with strongDiels-Alder dienophile reagents such as tetracyanoethylene, isderivatized to the hydro-dibenzo derivatives. However, it is clear that,as shown by these references, when acetylene is derivatized with moreweakly electron withdrawing groups and used as a Diels-Alder reagent,hydro-monobenzo derivatives are formed. Thus, there are obtaineddirectly from reaction of protoporphyrin with, for example dimethylacetylene dicarboxylate (DMAD), compounds shown as formulas 1 and 2 ofFIG. 1, wherein R¹ and R² represent the substituents on the originalacetylene-derived Diels-Alder reagent, R¹ C.tbd.CR² -- in this case,carbomethoxy. R¹ and R² are, generally, specifically carbalkoxy groupssuch as carbomethoxy or carboethoxy. R³ represents substituents presenton the porphyrin used in the reaction or substituents derived therefrom.In the Morgan reference, the reaction substrate was protoporphyrin-IXdimethyl ester; thus the ligand R³ was, in all cases,2-carbomethoxyethyl or 2-carboethoxyethyl.

The disclosed substituents in the Morgan and Pangka references for theacetylene-derived dienophile include phenylsulfonyl--i.e., SO₂ Ph,either as a single substituent, as described in the foregoing references(b-phenylsulfonylpropiate) or, putatively, wherein both R¹ and R² aresulfonyl derivatives. In general, R¹ and R² are each, independently,moderate electron-withdrawing substituents, and are, most commonly,carbalkoxy, or alkyl or aryl sulfonyl, or any other activatingsubstituents, which are not sufficiently electron-withdrawing to resultin reaction with both A and B rings rather than reaction with only one,such as cyano or --CONR⁵ CO-- wherein R⁵ is aryl or alkyl. One of R¹ andR² may optionally be H while the other is an electron withdrawingsubstituent of sufficient strength to facilitate the Diels-Alderreaction.

As used herein, carboxy is, as conventionally defined, --COOH andcarbalkoxy is --COOR, wherein R is alkyl; carboxyalkyl refers to thesubstituent --R'--COOH wherein R' is alkylene; carbalkoxyalkyl refers to--R'--COOR wherein R' and R are alkylene and alkyl respectively. Alkylis a saturated straight or branched chain hydrocarbyl of 1-6 carbonatoms such as methyl, n-hexyl, 2-methylpentyl, t-butyl, n-propyl, and soforth. Alkylene is as alkyl except that the group is divalent. Aryl oralkyl sulfonyl moieties have the formula SO₂ R wherein R is alkyl asabove-defined, or is aryl, wherein aryl is phenyl optionally substitutedwith 1-3 substituents independently selected from halo (fluoro, chloro,bromo or iodo), lower alkyl (1-4C) or lower alkoxy (1-4C). In addition,one or both R¹ of R² can itself be aryl--i.e., phenyl optionallysubstituted as above defined.

As shown in FIG. 1, the adduct formed by the reaction of R¹--C.tbd.C--R² with the protoporphyrin-IX ring system (R³ is a protectedform of 2-carboxyethyl such as 2-carbomethoxyethyl or2-carboethoxyethyl; R⁴ is CH═CH₂) are compounds of the formulas 1 and 2wherein the compound in formula 1 results from addition to the A ringand formula 2 results from addition to the B ring. In these resultingproducts of formulas 1 and 2, R⁴ remains CH═CH₂, however this vinylgroup is readily derivatized to other embodiments of R⁴ by addition toor oxidation of the vinyl ring substituent of ring B in formula 1 orring A in formula 2. The addition or oxidation products can be furthersubstituted if the added substituents are functional leaving groups--forexample --Br may be substituted by --OH, --OR (R is alkyl 1-6C asabove), or --NH₂, --NHR, --NR₂, etc. In preferred embodiments, one ofthe added substituents is hydrogen, and the other is selected from thegroup consisting of halo (fluoro, chloro, bromo or iodo), hydroxy, loweralkoxy, amino or an amide, sulfhydryl or an organo-sulfide or can be,itself, hydrogen. Addition to the vinyl group does not appreciablychange the absorption spectrum of the resulting compound. The product ofthe Markonikov addition of water provides a substituent structureanalogous to the hematoporphyrin ring system at the relevant ring. Thus,the compounds of the invention include various groups as R⁴, includingsubstituents which provide additional porphyrin or porphyrin-relatedring systems, as will be further described below.

R³ in protoporphyrin-IX is 2-carboxyethyl (--CH₂ CH₂ COOH). However, thenature of R³ (unless it contains a π-bond conjugated to ring π-bond), isordinarily not relevant to the progress of the Diels-Alder reaction orto the effectiveness and absorption spectrum of the resulting product.R³ can thus be, for example, lower alkyl (1-4C), or 1-carboxyalkyl(2-6C) or the esters or amides thereof. The R³ substituent may also besubstituted with halogen as above-defined, or with other nonreactivesubstituents. However, as the convenient starting materials for the Gpcompounds of the invention are the naturally occurring porphyrins, thepreferred substituents for R³ are CH₂ CH₂ COOH or --CH₂ CHR₂ COOR,wherein R is alkyl (1-6C).

It should be noted that while the nature of the R³ substituent does notordinarily influence the course of the Diels-Alder reaction by alteringthe nature of the diene substrate, derivatization may be necessary topromote the reaction by providing suitable solubility characteristics orto prevent interference with the reaction. Thus, the Diels-Alderreactions described by Morgan et al. and by Pangka et al. utilized thedimethylester of protoporphyrin-IX as a substrate in order to preventinterference with the reaction by the free carboxyl group and to providesuitable solubility characteristics.

In the BPD compounds of the invention, it has been found advantageous tohydrolyze or partially hydrolyze the esterified carboxy group in --CH₂CH₂ COOR. The hydrolysis occurs at a much faster rate than that of theester groups of R¹, R², and the solubility and biodistributioncharacteristics of the resulting compounds are more desirable than thoseof the unhydrolyzed form. Hydrolysis results in the diacid or monoacidproducts (or their salts).

The hydro-monobenzoporphyrins which directly result from the Diels-Alderreaction described in the cited references can also be isomerized astherein described (see Morgan et al. and Pangka et al., supra) tocompounds of formulas shown as 3 and 4 of FIG. 1 by treatment withsuitable reagents such as triethylamine (TEA) in methylene chloride or1,5-diaza bicyclo [5.4.0]undec-5-ene (DBU). The stereochemistry of theproduct is determined by the choice of reagent.

The depictions of compounds 3 and 4 in FIG. 1 do not show the relativeposition of the exocyclic methyl group (ring A of formula 3 and ring Bof formula 4) with respect to the R² substituent. It has been found bythese authors that rearrangement using TEA gives cis geometry for theangular methyl group and R², while treatment with DBU results in thetrans product. This cis product is evidently kinetically controlledsince treatment of the cis product with DBU results in a furtherrearrangement to trans stereochemistry. Thus, formulas 3 and 4 of FIG. 1show the rearranged products generically, from either TEA or DBUcatalyzed rearrangement in rings A and B respectively.

In addition, the Diels-Alder products can be selectively reduced bytreating with hydrogen in the presence of palladium on charcoal to givethe saturated ring analogs, shown as formulas 5 and 6 in FIG. 1,corresponding to the respective Diels-Alder products of rings A and B.These reduced products are less preferred embodiments, and are lessuseful in the method of the invention than the compounds of formulas1-4.

The description set forth above with respect to the compounds offormulas 1 and 2 concerning derivatization by conversion of theremaining vinyl substituent (R⁴) and with respect to variability of --R³applies as well to the compounds of formulas 3, 4, 5 and 6.

The compounds of formulas 3 and 4 (BPD), and especially those which havehydrolyzed and partially hydrolyzed carbalkoxy groups in R³, are mostpreferred. Compounds of the invention which contain --COOH may beprepared as the free acid or in the form of salts with organic orinorganic bases.

It will be noted that many of the compounds of FIG. 1 contain at leastone chiral center and therefore exist as optical isomers. The conjugatesand methods of the invention include compounds having bothconfigurations of the chiral carbons, whether the compounds are suppliedas isolates of a single stereoisomer or are mixtures of enantiomersand/or diastereomers. Separation of mixtures of diastereomers may beeffected by any conventional means; mixtures of enantiomers may beseparated by usual techniques of reacting them with optically activepreparations and separating the resulting diastereomers.

It should further be noted that the reaction products may be unseparatedmixtures of A and B ring additions, e.g., mixtures of formulas 1 and 2or 3 and 4 or 5 and 6. Either the separated forms--i.e., formula 3 aloneor 4 alone, or mixtures in any ratio may be employed in the methods oftherapy and diagnosis set forth herein.

The name "dihydro"-monobenzoporphyrin describes the direct andrearrangement products of the Diels-Alder reaction of the porphyrin ringsystem with R¹ C=C--R² --, "tetrahydro"-monobenzoporphyrin describes theforegoing reduced products of formulas 5 and 6, and"hexahydro"-monobenzoporphyrin describes the analogs containingexocyclic "benzo" ring completely reduced. Hydro-monobenzoporphyrin isused generically to include a three classes of oxidation state. Themonobenzo-porphyrins per se are outside the scope of the invention astheir absorption maxima do not fall within the required range.

FIG. 2 shows four particularly preferred compounds of the inventionwhich have not been previously described in the art. These compounds arecollectively designated benzoporphyrin derivative (BPD) as they areforms of Gp having the formula 3 or 4. These are hydrolyzed or partiallyhydrolyzed forms of the rearranged products of formula 3 and 4, whereinone both of the protected carboxyl groups of R³ are hydrolyzed. Theester groups at R¹ and R² hydrolyze relatively so slowly that conversionto the forms shown in FIG. 2 is easily effected.

For purposes of this description, R³ is --CH₂ CH₂ COOR^(3'). As shown inFIG. 2, each R^(3') is H in preferred compound BPD--DA, R¹ and R² arecarbalkoxy, and derivatization is at ring A; BPD--DB is thecorresponding compound wherein derivatization is at ring B. BPD--MArepresents the partially hydrolyzed form of BPD--DA, and BPD--MB, thepartially hydrolyzed form of BPD--DB. Thus, in these latter compounds,R¹ and R² are carbalkoxy, one R^(3') is H and the other R^(3') is alkyl(1-6C). The compounds of formulas BPD--MA and BPD--MB may be homogeneouswherein only the C ring carbalkoxyethyl or only the D ringcarbalkoxyethyl is hydrolyzed, or may be mixtures of the C and D ringsubstituent hydrolyzates. In addition, mixtures of any two or more ofBPD--MA, --MB, --DA and --DB may be employed in the method of theinvention.

As these hydrolyzed forms of the Diels-Alder product are previouslyundisclosed, the invention is also directed to these compounds. Thus, inanother aspect, the invention is directed to compounds of the formulasshown in FIG. 2 wherein R¹ and R² are as above defined, and R is alkyl(1-6C). Preferred are embodiments wherein R¹ and R² are carbalkoxy,especially carbomethoxy or carboethoxy.

Certain other embodiments wherein R⁴ is other than vinyl or wherein R³is a nonnative substituent are also not disclosed in the art and theinvention is directed to them, i.e., the invention is directed to thecompounds shown in FIG. 1 wherein

each R¹ and R² is independently selected from the group consisting ofcarbalkoxy (2-6C), alkyl (1-6C) sulfonyl, aryl (6-10C) sulfonyl, aryl(6-10C); cyano; and --CONR⁵ CO-- wherein R⁵ is aryl (6-10C) or alkyl(1-6C);

each R³ is independently carboxyalkyl (2-6C) or a salt, amide, ester oracylhydrazone thereof, or is alkyl (1-6C); and

R⁴ is CHCH₂, CHOR^(4'), --CHO, --COOR^(4'), CH(OR^(4')), CH₃,CH(OR^(4'))CH₂ OR^(4'), --CH(SR^(4'))CH₃, --CH(NR^(4') ₂)CH₃,--CH(CN)CH₃, --CH(COOR^(4'))CH₃, --CH(OOCR^(4'))CH₃, --CH(halo)CH₃, or--CH(halo)CH₂ (halo),

wherein R^(4') is H, alkyl (1-6C) optionally substituted with ahydrophilic substituent, or

wherein R⁴ is an organic group of <12C resulting from direct or indirectderivatization of vinyl, or

wherein R⁴ is a group containing 1-3 tetrapyrrole-type nuclei of theformula --L--P as herein define;

wherein when R⁴ is CHCH₂, both R³ cannot be 2-carbalkoxyethyl.

Compounds of the formulas 3 and 4 and mixtures thereof are particularlypreferred. Also preferred are those wherein R¹ and R² are the same andare carbalkoxy, especially carboethoxy; also preferred are those whereinR⁴ is --CHCH₂, CH(OH)CH₃ or --CH(halo) CH₃, or is a group containing 1-3tetrapyrrole-type nuclei of the formula --L--P (defined below).

As used herein, "tetrapyrrole-type nucleus " represents a four-ringsystem of the skeleton: ##STR1## and a salt, ester, amide oracylhydrazone thereof, which is highly conjugated. It includes theporphyrin system, which is, in effect, a completely conjugated system,the chlorin system, which is, in effect, a dihydro form of theporphyrin, and the reduced chlorin system, which is a tetrahydro form ofthe completely conjugated system. When "porphyrin" is specified, thecompletely conjugated system is indicated; Gp is effectively a dihydroform of the porphyrin system.

One group of compounds of the invention is that wherein the substituentR⁴ includes at least one additional tetrapyrrole-type nucleus. Theresulting compounds of the invention are dimers or oligomers in which atleast one of the tetrapyrrole-type ring systems is Gp. Linkage betweenthe Gp moiety through the position of R⁴ to an additionaltetrapyrrole-type ring system may be through an ether, amine or vinyllinkage. Additional derivatization in the case of porphyrin ring systemswhich have two available substituent positions (in both A and B rings)corresponding to R⁴ can also be formed, as further described below.

As stated above, the compounds of formulas shown in FIG. 1 include thosewherein the embodiment of R⁴ is formed by addition to the vinyl groupsof initial Gp products. Thus, R⁴ can be any substituent consistent withthat formed by a facile addition reaction. Thus, both added substituentscan be, for example, OH or halo, and these substituents can be furthersubstituted, or the addition reagent may be of the form HX wherein H isadded to the ring-adjacent carbon to provide R⁴ of the form ##STR2##

The vinyl group can also be oxidized to obtain R⁴ as CH₂ OH, --CHO, orCOOH and its salts and esters.

Thus, in general R⁴ represents any substituents to which the vinyl group--CH═CH₂ is readily converted by cleavage or addition, and furtherresultants of reaction of leaving groups with additional moieties.Typical R⁴ substituents include: ##STR3## CH(OH)Me, --CHBrMe,--CH(OMe)Me, --CH(pyridinum bromide)Me, --CH(SH)Me and the disulfidethereof, --CHOHCH₂ OH, --CHO, and --COOH or --COOMe.

When R⁴ is --L--P, the substituent formula "--L--P" represents asubstituent wherein --L-- is selected the group consisting of ##STR4##and P is selected from the group consisting of Gp wherein Gp is of theformula 1-6 shown in FIG. 1, but lacking R⁴ and conjugated through theposition shown in FIG. 1 as occupied by R⁴ to L, and a porphyrin of theformula ##STR5## wherein R³ and R⁴ are as above-defined, and theunoccupied bond is then conjugated to L. It is understood that theabbreviation ##STR6## represents a porphyrin of the formula: ##STR7##wherein each R is independently H or lower alkyl (1-4C).

(It is also understood that when --L-- is of the formula (e) or (f), thering system to which the double bond is attached will have a resonancesystem corresponding to ##STR8## in the ring to which the double bond isattached, as shown.)

Typical embodiments of --L--P include ##STR9## wherein R⁴ is as abovedefined. Thus, compounds of the invention include: ##STR10## and thelike.

Preparation of the Dimers and Oligomers

The dimers and oligomeric compounds of the invention can be preparedusing reactions analogous to those for dimerization and oligomerizationof porphyrins per se. The green porphyrins or green porphyrin/porphyrinlinkages can be made directly, or porphyrins may be conjugated, flowedby a Diels-Alder reaction of either or both terminal porphyrins toconvert to the corresponding green porphyrin.

For formation of compounds of the invention where --L-- is of theformula ##STR11## i.e., an ether linkage, the Gp vinyl group isconverted to the halide, preferably the chloride, by treating the Gp orporphyrin in a solution of, for example, methylene chloride with HBr torecover the addition product. The resulting product is harvested byevaporation in vacuo, redissolved in methylene chloride and added to aninsoluble base such as solid potassium carbonate. To this is added anequivalent of the tetrapyrrole-type nucleus "P" to be linked wherein thereactive R⁴ moiety of "P" is 1-hydroxyethyl. The mixture is stirred forthe appropriate amount of time, around 12 hours, generally, and theresulting diastereomeric pair of dimers (the enantiomeric paired formand a meso form) can be separated from the mixture chromatographically.The tetrapyrrole-type nucleus represented by "P" in this procedure canbe either another Gp or a porphyrin.

If the "P" substituent is a porphyrin, an additional vinyl group may bemade available for further halogenation and further reaction to formhigher order oligomers.

For embodiments wherein --L-- contains a vinyl group, the dimers areobtained by treating Gp or porphyrin wherein R⁴ is 1-hydroxyethyl withan equivalent amount of the linking tetrapyrrole-type nucleus alsohaving the linking R⁴ as 1-hydroxyethyl with a strong, nonnucleophilicacid, such as trifluoromethyl sulfonic acid. This treatment results inprecipitation of the resulting methylpropenyl linked dimer. (Theether-linked dimer can be formed as a side product in this reaction bysubstituting alternative acids such as sulfuric acid.)

The amino-linked compounds can be formed by treatment of the vinyl groupwith HBr followed by treatment with the appropriate amine to obtain thedesired linkage.

The Target-Specific Component

The target-specific component can be, for example, an immunoglobulin orportion thereof or a ligand specific for receptor.

The immunoglobulin component can be any of a variety of materials. Itmay be derived from polyclonal or monoclonal antibody preparations andmay contain whole antibodies or immunologically reactive fragments ofthese antibodies such as F(ab')₂, Fab, or Fab' fragments. Use of suchimmunologically reactive fragments as substitutes for whole antibodiesis well known in the art. See, for example Spiegelberg, H. L., in"Immunoassays in the clinical Laboratory" (1978) 3:1-23.

Polyclonal anti-sera are prepared in conventional ways by injecting asuitable mammal with antigen to which antibody is desired, assaying theantibody level in serum against the antigen, and preparing anti-serawhen the titers are high. Monoclonal antibody preparations may also beprepared conventionally such as by the method of Koehler and Milsteinusing peripheral blood lymphocytes or spleen cells from immunizedanimals and immortalizing these cells either by viral infection, byfusion with myelomas, or by other conventional procedures, and screeningfor production of the desired antibodies by isolated colonies. Formationof the fragments from either monoclonal or polyclonal preparations iseffected by conventional means as described by Spiegelberg, H. L.,supra.

Particularly useful antibodies exemplified herein include the monoclonalantibody preparation CAMAL-1 which can be prepared as described byMalcolm, A., et al., Ex Hematol (1984) 12:539-547; polyclonal ormonoclonal preparations of anti-M1 antibody as described by Mew, D., etal., J Immunol (1983) 130:1473-1477 (supra); and B16G antibody which isprepared as described by Maier, T., et al., J Immunol (1983) 131:1843;Steele, J. K., et al., Cell Immunol (1984) 90:303.

The foregoing list is exemplary and certainly not limiting; once thetarget tissue is known, antibody specific for this tissue may beprepared by conventional means. Therefore the invention is applicable toeffecting toxicity against any desired target.

The ligand specific for receptor, Re*, refers to a moiety which binds areceptor at cell surfaces, and thus contains contours and chargepatterns which are complementary to those of the receptor. The ligandspecific for receptor is symbolized in the formulas of the compounds ofthe invention as Re*, wherein the asterisk indicates that the moietybound in the compound of the invention is not the receptor itself, but asubstance complementary to it. It is well understood that a wide varietyof cell types have specific receptors designed to bind hormones, growthfactors, or neurotransmitters. However, while these embodiments ofligands specific for receptor are know and understood, the phrase"ligand specific for receptor," as used herein, refers to any substance,natural or synthetic, which binds specifically to a receptor.

Examples of such ligands include the steroid hormones, such asprogesterone, estrogens, androgens, and the adrenal cortical hormones;growth factors, such as epidermal growth factor, nerve growth factor,fibroblast growth factor, and so forth; other protein hormones, such ashuman growth hormone, parathyroid hormone, and so forth; andneurotransmitters, such as acetylcholine, serotonin, and dopamine. Anyanalog of these substances, such as agonists or antagonists, whichsucceeds in binding to the receptor is also included.

Linkage

The conjugation of the target-cell-specific component to thehydro-monobenzoporphyrin can be effected by any convenient means. Forproteins, such as Ig and certain Re*, a direct covalent bond betweenthese moieties may be effected, for example, using a dehydrating agentsuch as a carbodiimide, in which case L represents a covalent bond. Aparticularly preferred method of covalently bindinghydro-monobenzoporphyrins to the immunoglobulin moiety is treatment with1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDCI) in the presenceof a reaction medium consisting essentially of dimethyl sulfoxide(DMSO). A preparation using this preferred procedure is illustrated inExample 3 below.

Of course, other dehydrating agents such as dicyclohexylcarbodiimide ordiethylcarbodiimide could also be used as well as conventional aqueousand partially aqueous media.

Nonprotein receptor ligands can be conjugated to the Gp according totheir relevant functional groups by means known in the art.

The active moieties of the conjugate may also be conjugated throughlinker compounds which are bifunctional, and are capable of covalentlybinding each of the two active components. A large variety of theselinkers is commercially available, and a typical list would includethose found, for example, in the catalog of the Pierce Chemical Co.These linkers are either homo or heterobifunctional moieties and includefunctionalities capable of forming disulfides, amides, hydrazones, and awide variety of other linkages.

Other linkers include polymers such as polyamines, polyethers, polyaminealcohols, derivatized to the components by means of ketones, acids,aldehydes, isocyanates, or a variety of other groups.

The techniques employed in conjugating the active moieties of theconjugate include any standard means and the method for conjugation doesnot form part of the invention. Therefore, any effective technique knownin the art to produce such conjugates falls within the scope of theinvention, and the linker moiety is accordingly broadly defined only asbeing either a covalent bond or any linker moiety available in the artor derivable therefrom using standard techniques.

Label

For use in the method of the invention either the green porphyrincompounds per se or the conjugates may be further derivatized to acompound or ion which labels the drug. A wide variety of labelingmoieties can be used, including radioisotopes, chromophores, andfluorescent labels. Radioisotope labeling is preferred, as it can bereadily detected in vivo.

The compounds which are Gp alone or are conjugates of Gp with a specificbinding substance can be labeled with radioisotopes by coordination of asuitable radioactive cation in the porphyrin system. Useful cationsinclude technetium, gallium, and indium. In the conjugates, either orboth the specific binding substances can be linked to or associated withlabel, or the label can be conjugated or coordinated with the Gp moietyitself.

Metal Ions

The compounds of the invention can be administered or used in in vitromethods as shown above or when complexed to appropriate metal ions. Asis generally understood in the art, the tetrapyrrole-type nucleus can betreated with an appropriate ion such as magnesium ion, zinc ion,stannous ion, and the like to obtain the metal complex. As stated above,the metal ion may also be a radiolabel. The nature and desirability ofthe inclusion of a metal ion in the tetrapyrrole-type nucleus depends onthe specific application for which the compound is intended. When theinclusion of a metal ion is desired, the desired metal ion can beinserted using the appropriate metal salts under known conditions. Forexample, zinc ion can be introduced by treating the compound with zincacetate in 1:1 methylene chloride:methanol.

Administration and Use

The improved photosensitizing compounds of the invention are thus usefulin general, in the manner known in the art for hematoporphyrinderivative and for DHE. These materials are useful in sensitizingneoplastic cells or other abnormal tissue including infectious agents todestruction by irradiation using, preferably, visible light. Uponphotoactivation, the compounds seem to have no direct effect; the energyof photoactivation is believed to be transferred to endogenous oxygen toconvert it to singlet oxygen. This singlet oxygen is thought to beresponsible for the cytotoxic effect. In addition, the inventioncompounds photoactivated using appropriate excitation wavelengthsfluoresce. This fluorescence can be used to localize the tumor or othertarget tissue.

In general, the same wavelength range can be used for inducingcytotoxicity as for exciting fluorescence; if fluorescence is to bedirectly observed, however, it is advantageous to use appreciablyshorter wavelengths so that the excitation radiation does not interferewith the observation of the fluorescence. However, if a suitablyarranged apparatus is employed, the excitation wavelength can be quiteclose to the fluorescent wavelength. In addition, although all of thegreen porphyrin compounds of the invention can be activated by light inthe wavelength range of 670-780 nm, shorter wavelengths can also beused, if desired, for convenience. In many cases, the absorption spectrashow appreciable absorption down to the low 600 nm range as well as atappreciably shorter wavelengths.

The radiation for mediating cytotoxicity or fluorescence emission can besupplied by standard sources of visible radiation, includingincandescent or fluorescent light sources using suitable filters, or canbe supplied by photodiodes, such as light-emitting diodes at a narrowwavelength range. In addition, laser light is often convenient for thein situ delivery of light to the localized photosensitizer of theinvention. Thus, among the sources that have been used in photodynamictherapy and diagnosis in general are quartz, halogen and arc lampsources, monochromatic light from a fixed wavelength, gold vapor,tunable argon-pump dye laser or other wavelength-specific lasers, andstandard visible light sources in general.

Particularly preferred in the therapeutic and diagnostic practice of theinvention are light-emitting diodes (LEDs) which produce sufficientradiation to activate the photosensitizing compounds and are relativelyinexpensive, small in size, and do not require special utilities foroperation. The relatively broad-band light generated from LEDs, ascompared to the single wavelength generated by laser radiation, allowsadvantage to be taken of the broad wavelength absorption of theinvention compounds. BPD-MA, for example, has an absorption peak withsubstantial extinction coefficients extending from 680 nm to 700 nm. TheLEDs also contribute less heat to the irradiated biological tissue thando typical filtered broad-band light sources. LEDs are particularlyadaptable in cases where relatively large irradiation areas are requiredas in treatment, for example, of large superficial lesions of skincancer.

Most LEDs have emission bands of about 20-40 nm and can operate from thegreen (500 nm) to the near infrared; the LEDs appropriate for thecompounds of the invention generally will emit in the range of 600 nm to800 nm. Multiple LEDs positioned together can generate radiant fluxlevels of 40 mW/cm² to 100 mW/cm² and have operating lifetimes of100,000 hours or more. As these are solid state devices, there are noseparate components or moving mechanisms, nor is there requirement formaintenance other than replacement. They use low voltages and typicallyrequire no temperature regulation.

Typical lasers used in photodynamic therapy and diagnosis involving thecompounds of the invention include metal vapor or dye lasers, such asthe argon-pumped dye laser or copper vapor-pumped dye laser orNd:YAG-pumped dye laser, among others. In use, the laser systemgenerally consists of 2-3 separate lasers arranged serially to achievethe desired output wavelength and optical power. Lasers are lessefficient in conversion of electrical to optical energy than LEDs--forthe argon laser values in the range of 0.01-0.25% are typical--whereasLEDs have electrical-to-light conversion efficiencies of about 8%. LEDsalso have longer lifetimes as compared to the 2,000-3,000 hoursavailable from typical argon lasers.

Any suitable light source can be used for irradiation of the tissue toeffect cytotoxicity or excite fluorescence in those tissues in which theinvention compounds reside. However, light-emitting diodes arepreferred.

It is also feasible to generate the photoactivating light using a systemwhich produces light by virtue of a chemical reaction. In these systems,a chemical transition which liberates energy sufficient to excitevisible wavelength emissions from a suitable compound is responsible forthe radiation. A chemiluminescent system (CLS) wherein a substitutedoxamide reacts with hydrogen peroxide in the presence of a sulfonatedrubene to produce an intense yellow-red light lasting 10-20 minutes wasreported to be useful as an irradiation source in photodynamic therapyby Phillip, M. J., et al., in "Porphyrin Localization in Treatment ofTumors" (1984), Alan R. Liss, Inc., pp. 563-569; Phillip, M. J., et al.,Oncology (1989) 46:266-272. The CLS is injected directly into the targettumor after the photosensitizer has homed to the target. This CLS, aswell as alternative nontoxic light-generating systems, can be used asirradiation sources in the methods of the invention.

In addition to irradiation for excitation of the invention compounds fortherapy, additional forms of irradiation which can independently destroythe tissue irradiated can be used to supplement the effect of thephotodynamic therapy per se. The use of X-irradiation orgamma-irradiation as a direct treatment for tumors has been known formany years, and the combination of X-ray therapy with photodynamictherapy using art-known forms of porphyrins has been well documented,for example by Schwartz, S., et al., U Minn Med Bull (1955) 27:1-37. Aretrospective of this combination treatment was given by Dr. Schwartz atthe Third Biennial Meeting of the Third International PhotodynamicAssociation, Jul. 18, 1990, in Buffalo, N.Y. Thus, the therapeuticmethods of the invention can employ a variety of irradiation means foractivation of the photosensitizer, alone or in combination withadditional radiation designed for direct treatment of tumor, said directtreatment radiation typically including X-rays, microwave radiation, andthe use of additional photochemicals as chemiluminescent or fluorescentradiation transfer materials.

In addition to additional irradiation, the photodynamic treatment can beaccompanied by adjuvant therapy using approaches such as surgery andchemotherapy. Also, PDT potentiators, such as glucose, which depressestumor pH and results in greater accumulation of the photosensitizer andthus more effective cytotoxicity, can be used. This is suggested byThomas and Girotti, Photochem. Photobiol. (1989) 49:241-247. Otheradjuvant treatments which can be used along with photodynamic therapyinclude the use of protective agents such as cadmium chloride fortopical application, misonidazole (MISO), or ethanidazole for protectionagainst direct cellular phototoxicity of intermediate oxygenconcentration, or antiinflammatories such as ibuprofen and ASA asprotective agents. Additional inhibitors of PDT which can regulate itseffect include noradrenaline, propanalol, hydrazine, andphenoxybenzamine.

Typical of the indications targeted for photodynamic treatment includein vivo treatment for destruction of tumor tissue in solid tumors andfor dissolution of plaques in blood vessels (see, e.g., U.S. Pat. No.4,512,762); prevention of restenosis; and treatment of topicalconditions such as acne; athlete's foot; warts; papilloma, includingvenereal, laryngeal and dermal; unwanted tissue in general, such as hairfollicles or fat deposits; port wine stains; hypervascularization,including varicose veins and spider veins; and psoriasis. Otherindications include the systemic treatment of tumors and neoplastictissues, such as malignancies that occur in brain, face, mouth, throat,lung, gastric, rectal, prostate, ovarian, breast, skin (basal,melanoma), bone, blood, hematopoietic, lymph, bronchial, cervical,esophageal or colon tissues and Kaposi's Sarcoma. The inventioncompounds are also useful for treatment of biological products (such asblood for transfusion) for infectious agents, since the presence of amembrane in such agents promotes the accumulation of the drug.

In particular, the invention compounds are useful for eradicatinginfectious agents, in vivo or ex vivo, including viral contaminantsoften found in donated blood or blood products. Such infectious andviral contaminants include, for example, bacterial, fungal or parasiticinfection, hepatitis B, hepatitis A or hepatitis C virus, humanimmunodeficiency virus (HIV), cytomegalovirus (CMV), and Epstein-Barrvirus. Vesicular stomatitis virus (VSV), while not usually found inhuman blood, behaves in a similar manner in response to the photodynamictreatment. In addition, parasites such as Trypanosomes or Plasmodium aresusceptible targets. All of the foregoing infectious agents can beeradicated by the methods of the invention both in vivo and ex vivo.

For use in in vivo treatment or diagnosis of atherosclerotic plaques ormalignancies or infections treated systemically, the compounds of theinvention are injected, typically by intravenous injection, andpermitted sufficient time to home to the atherosclerotic plaques,malignancies or infective agents, usually about 30 minutes to 3 hours.The plaques or malignancies are then subjected to irradiation fortherapeutic effect to dissolve the plaque or destroy the tumor cells.Typical dosages are in the range of 100 μg/kg to 10 mg/kg of the drugbased on the weight of the subject. Due to the accumulation of the drugin the plaques or tumor cells, treatment is effected. As infectiousagents are distributed, focus of the radiation on the target can beeffected only using whole body radiation.

The hydro-monobenzoporphyrins are formulated into pharmaceuticalcompositions for administration to the subject or applied to an in vitrotarget using techniques known in the art generally. A summary of suchpharmaceutical compositions may be found, for example in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latestedition.

The conjugates and hydro-monobenzoporphyrins of the present invention,labeled or unlabeled, can be administered systemically, in particular byinjection, or can be used topically. The Gp or conjugates can be usedsingly or as components of mixtures.

Injection may be intravenous, subcutaneous, intramuscular, or evenintraperitoneal. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid form suitable forsolution or suspension in liquid prior to injection, or as emulsions.Suitable excipients are, for example, water, saline, dextrose, glyceroland the like. Of course, these compositions may also contain minoramounts of nontoxic, auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and so forth.

Systemic administration can also be implemented through implantation ofa slow release or sustained release system, by suppository, or, ifproperly formulated, orally. Formulations for these modes ofadministration are well known in the art, and a summary of such methodsmay be found, for example, in Remington's Pharmaceutical Sciences(supra).

If treatment is to be localized, such as for the treatment ofsuperficial tumors or skin disorders, the active conjugates or compoundsmay be topically administered using standard topical compositionsinvolving lotions, suspension, or pastes. The topical formulations willcontain typical excipients and are in the form of liquids, creams, gelsor ointments. These formulations may also contain penetrants, such asDMSO and/or additional ingredients which affect depth of administrationlocally.

The quantity of conjugates or green porphyrin derivative to beadministered depends on the choice of active ingredient, the conditionto be treated, the mode of administration, the individual subject, andthe judgement of the practitioner. Depending on the specificity of thepreparation, smaller or larger doses may be needed. For compositionswhich are highly specific to target tissues, such as those whichcomprise conjugates of the green porphyrin with a highly specificmonoclonal immunoglobulin preparation or specific receptor ligand,dosages in the range of 0.05-1 mg/kg are suggested. For compositionswhich are less specific to the target tissue, larger doses, up to 1-10mg/kg may be needed. The foregoing ranges are merely suggestive, as thenumber of variables in regard to an individual treatment regime is largeand considerable excursions from these recommended values are expected.

In addition to in vivo use, the compounds of the invention can be usedin the treatment of materials in vitro to destroy harmful viruses orinfectious agents. For example, blood plasma or blood which is to beused for transfusion or banked for future transfusion can be treatedwith the compounds of the invention and irradiated to effectsterilization. In addition, biological products such as Factor VIIIwhich are prepared from biological fluids can be irradiated in thepresence of the compounds of the invention to destroy contaminants. Thephotosensitizer is added to the sample in a concentration rangedependent on the nature of the sample, but generally, for example, forblood amounts of 0.5-10 μg/ml of the photosensitizer are used. Thebiological sample is incubated with the photosensitizer for one toseveral hours, usually at room temperature, before exposure to light.The biological fluid in the presence of photosensitizer is thenirradiated using an appropriate light source, such as a tungsten lightbulb emitting over the range of the visible spectrum with lightintensity of about 5-10 mW/cm², or LEDs with emission bands in the600-800 nm range. The light from broad-spectrum sources is filtered soas to deliver the appropriate wavelength for absorption by thephotosensitizer.

For use as a diagnostic in localizing tumor tissue or in localizingplaques, the compounds or conjugates of the invention are administeredsystemically in the same general manner as described above with respectto photodynamic therapy. The waiting period to allow the drugs to clearfrom tissues to which they do not home is approximately the same, about30 minutes to 10 hours. After the compounds of the invention or theirconjugates have been permitted to home, the location of the targettissue is determined by detecting the presence of the drug.

For diagnosis, the compounds or conjugates may be used along with, ormay be labeled with, a radioisotope or other detecting means. If this isthe case, the detection means depends on the nature of the label.Scintigraphic labels such as technetium or indium can be determinedusing ex vivo scanners. Specific fluorescent labels can also be used,but these require prior irradiation, as does the detection based onfluorescence of the compounds of the invention themselves.

For activation of the fluorescence of the compounds of the invention,any suitable absorption wavelength is used. This can be supplied usingthe various methods described above to provide cytotoxicity. Suitabledetection means for the emitted visible radiation are then arranged, asexemplified, for example, in Example 15 hereinbelow. In a typicalprotocol, several hours before irradiation, approximately 0.5 mg/kg ofthe green porphyrin is injected intravenously and then excited by awavelength of, for example, 630 nm, and the fluorescence is then readat, for example, 690 nm. Suitable devices for the detection offluorescence are set forth in U.S. Pat. No. 4,649,151 and U.S. Ser. Nos.502,447 filed Mar. 30, 1990 and 572,902 filed Aug. 24, 1990, which areincorporated herein by reference. Detection and localization of plaquesor tumors using the compounds of the invention can be combined withother detection methods, if desired. For example, ultrasound has beenused for the detection of various tumors in situ (Dalla Palma, L., etal., Acta Oncologica (1989) 28:157-162).

EXAMPLES

The following examples are intended to illustrate the invention but notto limit its scope.

EXAMPLE 1 In Vitro Photosensitization by Green Porphyrins

Target cells were washed three times in serum-free medium (DME), countedand made up to a concentration of 10⁷ cells per ml.

For the "affinity" assay, in the dark, 100 μl of the target cellsuspension and 100 μl of the test or control compound were mixed."Labeling" was allowed to continue for one hour at 4° C., and labeledcells were washed in the dark three times with 3 ml medium each time andresuspended in fresh medium. The resuspended cells were then subjectedto light exposure at 300-750 nanometers for 30 minutes.

In a "direct" assay the target cells were irradiated immediately uponaddition of the test or control compound.

The effect of irradiation was estimated using methods appropriate to thetarget cells.

When human erythrocytes (RBCs) were used as target cells, the hemolysiscaused by irradiation of control (hematoporphyrin, Hp) labeled and greenporphyrin (Gp) labeled cells were estimated visually. The Gp used inthis Example was the BPD-DB of FIG. 2 wherein R¹ and R² are carboethoxy.Repeated tests showed this green porphyrin to be 20-30 times more activethan Hp in this assay. Thus, a concentration of 250 ng/ml Hp wasrequired under the above conditions to obtain 50% hemolysis while only10 ng/ml of green porphyrin was required to hemolyze 50% of the RBCs.

When the murine mastocytoma cell line P815 was used, the results weredetermined as follows:

The cells were labeled as above using concentration of 10-50 ng/ml of Hpas control and the BPD-DB as the test substance. The resuspended cellswere treated with 300-750 nm light for 30 minutes and the viabilityresulting was estimated by direct counting using eosin-Y exclusion, astandard procedure for differentiating living from dead cells.

In other determinations conducted as above, the cells recovered fromlight exposure were assayed for viability by incubating them for 18hours in 10 μCi/ml tritium-labeled thymidine according to the standardprocedure whereby thymidine incorporation is equated with viability. Thecells were harvested and radioactivity uptake was measured by ascintillation counter.

Fifty percent of the P815 cells were killed at 580 ng/ml Hp, but only 32ng/ml green porphyring (as BPD-DB) was required.

The results of each determination on a variety of cells is shown inTable 1(LD₅₀ in the concentration of compound required to kill 50% ofthe cell population.)

                  TABLE 1                                                         ______________________________________                                                      LD.sub.50 (ng/ml)                                                             Direct test    Affinity test                                    Cell line       Gp      Hp       Gp   Hp                                      ______________________________________                                        Normal lymphocytes                                                                            4.2      31      11     100                                   HL-60           3.5      64      7.2    145                                   K562            70      770      33   2,500                                   KG-1            163     960      80   2,350                                   P815            32      580      26   1,300                                   ______________________________________                                    

EXAMPLE 2 Selective Binding of Green Porphyring

P815 cells were incubated as described in Example 1 using 1-200 ng/ml Hpor Gp. The Gp was BPD-DB of FIG. 2 wherein R¹ and R² are carboethoxy.The cells were labeled in the dark for 30 minutes, washed free ofunabsorbed porphyrins, resuspended, and then exposed to 300-750 nm lightfor another 30 minutes. Viability of the cells was established bytritiated thymidine incorporation after labeling with 20 μCi/mltritiated thymidine and incuating at 37° C. for 18 hours.

The results showed that 50% of the P815 cells were destroyed at 6-20ng/ml BPD-DB or at 200 ng/ml hematoporphyrin.

EXAMPLE 3 Preparation of Immunoconjugates

This example describes methods of preparation for immunoconjugates offour different antibody preparations with either hematoporphyrin (Hp) orgreen porphyrin (Gp); in this example, Gp is BPD-DB of FIG. 2 wherein R¹and R² are carboethoxy. The antibodies employed were CAMLA-1, anti-M1antibody, and B16G antibody, all prepared as described hereinabove, andaffinity purified rabbit/anti-mouse Ig (RαMIg). In addition, a purifiedirrelevant monoclonal preparation (C-MAb) was used where a control wasdesired.

One preparation of the conjugates is basically as described in Mew, D.,et al., J Immunol (1983) 130:1473 (supra). Briefly, the 220 mg pH 0.2HCl (Sigma Chemical Co., St. Louis, Mo.) in 25 ml water and 0.8 mlN,N-dimethylformamide was added 20 mg 1-ethyl3-(3-dimethylaminopropyl)-carbodiimide HCl (EDCI) in 0.6 ml water. After30 minutes, this solution was mixed with 15 mg of the antibody proteindissolved in 5 ml distilled water and incubated for 5 hours. During thisperiod, the pH of the solution was monitored and adjusted to between 6and 7. Then 50 μl of monoethanolamine were added, and the solution wasallowed to stand overnight at room temperature. The solution wasdialyzed against 0.001M phosphate buffer pH 7.4 for four days with threechanges per day and overnight against PBS. The conjugate of greenporphyrin is analogously prepared.

In a preferred method, the conjugation is conducted in an entirelynonaqueous solvent.

In a typical protocol, 2 ml of a dispersion in DMSO containing 5 mg eachof the Hp or Gp and the dehydrating agent is prepared and stirred for 30minutes at room temperature under nitrogen. To this is added adispersion containing 2 mg of the appropriate immunoglobulin in 2 ml ofDMSO, and the resulting mixture stirred for another 10 minutes. Thismixture is then worked up by dilution in phosphate-buffered saline, pH7.4 (PBS) by adding 5 times the volume of PBS containing 50 μlmonoethanolamine, and is then dialyzed against PBS using three changesof wash.

Alternatively, 2 ml of a dispersion containing 5 mg each of Hp or Gp, alinking agent, and a dehydrating agent is prepared and stirred forapproximately 15 minutes at room temperature under nitrogen. To this isthen added a dispersion containing about 2 mg of the immunospecificprotein in 2 ml of tetrahydrofuran and the resulting mixture stirred foranother 10 minutes. The mixture is then worked up as described above.

The foregoing procedures are appropriate for CAMAL-1 and for theremaining antibody preparations above listed.

In addition, the following preparations were made specifically with B16Gand RαMIg:

B16G

11 mg of hematoporphyrin plus 11 mg EDCI in 4 ml spectral grade DMSO wasstirred for 30 minutes under nitrogen at room temperature before theaddition of 20 mg lyophilized B16G antibodies, prepared as described byMaier, T., et al., J Immunol (1983) 131:1843, in 2 ml DMSO. Theresulting mixture was stirred for 40 seconds at room temperature andworked up as described above. The resulting product contained 375 mgHp/mg B16G. A similar procedure is used substituting Gp for Hp.

RαMIg

400 mg of EDCI and 400 mg hematoporphyrin in 1 DMSO were stirred for 30minutes under nitrogen at room temperature as above before the additionof 800 μg lyophilized RαMIg antibodies, prepared as described by Mew,D., et al., J Immunol (1983) 1473-1477, in 1 ml DMSO. The resultingmixture was stirred for 30 seconds and worked up as described above toobtain a product containing 200 μg Hp/mg RαMIg. A similar procedure isused substituting Gp for Hp.

EXAMPLE 4 Specificity of Immunoconjugates in Vitro

In the following determinations, the levels of antibody conjugation wereas follows, expressed as μg Hp or green porphyrin (Gp) per mgimmunoglobulin:

RαMIg-Hp: 110 μg/mg;

B16G-Hp, 156 μg/mg;

CAMAL-1-Hp, 260 μg/mg;

Anti-M1-Hp, 170 μg/mg;

C-MAb-Hp, 95 μg/mg;

RαMIg-Gp, 120 μg/mg;

B16G-Gp, 165 μg/mg;

CAMAL-1-Gp, 75 μg/mg;

C-MAb-Gp 90 μg/mg.

The Ig-Hp and Ig-Gp conjugates are tested against cells in vivo bymixing the conjugates with the appropriate cell types, along withsuitable controls, and then exposing the labeled cells to irradiation.Procedures for carrying out this assay were described in detail in Mew,D., et al., Cancer Research (1985), for CAMAL-1, and by Mew, D., et al.,J Immunol (1983), for Anti-M1, both references cited hereinabove andincorporated herein by reference.

Briefly, for CAMAL-1, three cell lines, WC4, WC6 and WC2 (WC4 and WC6produces the CAMAL antigen, but WC2 does not), are labeled with theappropriate Ig-Hp or Ig-Gp preparation as described above in Example 1.The labeled cell preparations containing 10⁶ cells each are introducedto Rose chambers and exposed to light activation with a laser at 630 nm.The results for various preparations are then compiled.

For the anti-M1 conjugate, M1 tumor cells are used as target cells andtreated with the Ig-Hp, Ig-Gp conjugates or drug or antibody alone orthe combination of antibody and drug, but uncoupled, by incubating themin 6% CO₂ humidified incubator at 37° for two hours. The cells arewashed three times in PBS and then plated and exposed to fluorescentlight overnight. The cells are assessed for viability by tritiatedthymidine uptake as above.

For the B16G conjugates, A10, P815, and L1210 cells are used as targetcells. (A10 cells are a T-cell hybridoma which secretes a B16G-reactiveT suppressor factor; P815 cells are also reactive with B16G.) The invitro study is done using a direct method employing the B16G-Hp orB16G-Gp conjugate or indirectly using unlabeled B16G antibodies andlabeled RαMIg-Hp or RαMIg-Gp.

In a direct method, 5×10⁵ cells are suspended in 1 ml DME/Hepescontaining the appropriate Ig-drug conjugate as test or control at Hp orGp concentrations of 320, 160, 80, 40 and 20 ng drug/ml. The cells areincubated in the dark at 37° for one hour, then washed three times in 5ml DME/Hepes and then resuspended in 1 ml of the same buffer. Three 100μl test portions of the labeled preparations are dispensed into flatbottom microtiter wells and the remainder of the cell suspensions (700μl) are exposed to incandescent light (22.5 mW/cm²) at a distance of 20cm for one hour. Then three additional 100 ml aliquots are removed tomicrotiter wells. Tritium-labeled thymidine diluted in DME/Hepescontaining 20% FCS is then added to all microtiter wells in 100 mlaliquots so that 2 μCi of labeled thymidine is added to each well.Cultures are incubated for 18 hours at 37° C. and humidified 10% CO₂ andthen harvested on a MASH harvester. Thymidine incorporation was measuredwith an Hp scintillation counter (Tri-Carb Model 4550). The results ofthis study for Ig-Hp are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        B16G Hp    % killing of cell lines                                            (ng Hp/ml) A10          P815   L1210                                          ______________________________________                                        320        100          70     55                                             160        100          50     10                                             80         100          20     0                                              40         65           10     0                                              20         20            0     0                                              ______________________________________                                        C-Mab-Hp                                                                      (ng Hp/ml) A10          P815   L1210                                          ______________________________________                                        320        63           75     50                                             160        35           48     15                                             80          0           25     0                                              40          0           12     0                                              20          0            0     0                                              ______________________________________                                    

In an indirect assay, the A10 suspended cells, prepared as describedabove, are exposed to 50 μg/ml of either B16G or a control antibodyC-Mab at 4° C. for 30 minutes, washed in DME/Hepes, and then exposed foran additional 30 minutes at 4° C. in the dark to varying concentrationsof RαMIg-Hp or RαMIg-Gp between 2 μg/ml and 15 ng/ml of Hp or Gp. Thecells are assessed for viability using labeled thymidine uptake asdescribed above. These results for Ig-Hp are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        RαMIg-Hp  Primary antibody                                              (ng/ml)         B16G    C-MAb                                                 ______________________________________                                        500             100     30                                                    250             85      22                                                    125             75      5                                                       52.5          60      2                                                       31.2          47      3                                                       15.6          18      1.5                                                   ______________________________________                                    

Similar results are obtained using corresponding conjugates with Gp.

EXAMPLE 5 In Vivo Cytotoxicity of the Immunoconjugates

The efficacy of the conjugates and of the Gp compounds of the inventionin vivo is also assessed. For the CAMAL-1 and anti-M1 conjugates, theprocedures are as described in the two Mew et al. papers referencedabove in Example 4. The Gp compound alone shows superior results atappropriate wavelengths as compared to the Hp labeled conjugates.

For the B16G-Hp or B16G-Gp conjugates and for the Gp (BPD-DB) alone, thein vivo studies are conducted as follows:

The in vivo test relies on the indirect effect of a population ofT-suppressor cells on tumors, which then serve as means to assess theeffectiveness of the irradiation treatment. P815 mastocytoma cells grownin syngeneic DBA/2 mice stimulate T-suppressor cells specific for thetumor. These T-suppressor cells impede the development of specificT-killer cells which would otherwise aid in the regression of the tumor.The T-cell hybridoma designated A10 above secretes a T-suppressor factorwhich is associated with these T-suppressor cells. Thus, selectivekilling of these T-suppressor cell populations through reaction withconjugates in which the Ig is an antibody specific for the T-suppressorfactor on the surface of the cells (namely B16G) should result in tumorregression in mice bearing the P815 tumors.

Therefore, in this assay, DBA/2 mice are injected in the right flanksubcutaneously with 10⁴ P815 cells to incorporate the tumor. On dayeight, when the tumors are palpable (approx. 25-42 sq mm) the mice arerandomly sorted into groups of eight and injected IV with 150 μl PBScontaining nothing, Hp or Gp, B16G-Hp or B16G-Gp, B16G plus either drug,B16G alone or C-MAbHp or C-MAb-Gp. The levels of Hp are 50 μg per animalin all cases and B16G 310 μg in all cases (where appropriate).

The animals are maintained in the dark for two hours and then exposed tostrong light at 300-750 nm and 22.5 mW/cm². The animals were thentreated normally and monitored on a daily basis.

Animals treated with B16G Hp survived and were tumor free after 100days. Results obtained are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                           Mean sur-        % tumor-free                                                 vival time No. of                                                                              after 100                                 Experiment                                                                            Treatment  (days)     cures days                                      ______________________________________                                        1       PBS        25.0       0/7   0                                                 B16G-Hp    41.3       3/9   33                                        2       PBS        23.5       0/6   0                                                 Hp         21.0       0/8   0                                                 B16G-Hp    24.2       3/8   37.5                                      3       PBS        24.1       0/7   0                                                 Hp         23.4       0/7   0                                                 B16G + Hp  23.5       0/6   0                                                 B16G-Hp    29.2       2/7   29                                        4       PBS        25.2       0/8   0                                                 B16G       28.3       0/8   0                                                 Hp         24.2       0/8   0                                                 B16G + Hp  24.6       0/7   0                                                 B16G-Hp    36.7       3/7   43                                        5       PBS        23.8       0/8   0                                                 Hp         27.0       0/8   0                                                 C-MAb-Hp   20.3       0/8   0                                                 B16G-Hp    34.0       1/8   12.5                                      ______________________________________                                    

Similar results are obtained for Gp alone or Gp conjugates.

EXAMPLE 6 In Vitro Evaluation of BPD-DA', -MA, -DB and -MB

The four compounds shown in FIG. 2, wherein R¹ and R² are carbomethoxy,were tested in vitro as described in Example 1. All four compounds werephotosensitive; the monoacid forms BPD-MA and BPD-MB were somewhat moreactive.

EXAMPLE 7 Biodistribution and Degradation

Biodistribution studies have been conducted using tritiated BPD-MA andBPD-MB. Table 5 shows the ratios between ³ H-BPD-MA concentration in thetumor and in normal tissues determined at various times post-injectionin mice bearing P815 tumor as the average for 3 mice.

                  TABLE 5                                                         ______________________________________                                        Time Post Injection                                                           Tissue  3 h    24 h     48 h 72 h    96 h 168 h                               ______________________________________                                        Blood   0.52   1.45     1.37 1.66    2.77 3.65                                Brain   3.76   3.06     2.92 2.69    4.18 6.91                                Heart   1.09   1.71     1.63 1.46    2.24 2.51                                Intestine                                                                             2.42   1.85     1.88 1.48    3.29 2.23                                Lung    0.79   1.55     1.47 1.16    1.63 1.79                                Muscle  2.68   2.98     2.77 2.16    3.45 4.23                                Skin    2.57   1.64     1.95 1.57    2.03 3.51                                Stomach 1.57   1.89     2.08 2.04    2.23 2.98                                ______________________________________                                    

Tumor skin ratios are most favorable 3 hours after IV administration ofthe drug.

To determine biodegradability, tritiated BPD-MA was injected IV intoP815 tumor-bearing mice. The mice were sacrificed at either 3 or 24hours following injection and tumors, livers and kidneys were removed.The BPD-MA in these tissues was extracted and photoactivity was assessedin P815 target cells as described above in Example 1 under standard invitro conditions. While 100% of BPD-MA in tumor was active at 3 hours,only 39% was active at 24 hours; both the liver and kidney degraded BPDmore rapidly than did tumor tissue. Administration of tritiated BPD-MBin the same system gave similar results.

Similar studies using BPD-MA conjugated to an anti-keratin Mab in amodel murine system carrying the KLN squamous tumor cell line showedimproved concentration of the drug in the target tissue.

EXAMPLE 8 In Vivo Photosensitization by BPD

Studies of potential photosensitizers were performed using the M-1rhabdomycoscercoma system in DBA/J2 mice. The compositions to be testedwere diluted to a concentration of 800 mg/ml in PBS from a stocksolution in DMSO at 8 mg/ml (except Photofrin® II, which was diluteddirectly from the clinical vial). Animals (8 per group) received 0.1 ml(80 mg) of material IV 24 h prior to exposure to light, provided by a150 W tungsten bulb, red filter (transmits light >600 nm), hot mirror(reflects light >720 nm) and 2 fiber optics, at 567 Jo/cm².

The results, shown in Table 6, indicate all BPD compounds tested gavepositive results. The superior results shown by Photofrin® IIcompositions are explainable by the observation that initial tumor sizeswere smaller (a result of chance).

                  TABLE 6                                                         ______________________________________                                                                        Tumor Volume at                                         Days Tumor Number of  Time of Light                                 Photosensitizer                                                                         Free (PR)  Cures*     Treatment (mm.sup.3)                          ______________________________________                                        None      0.5        2          22.4 ± 7.8                                 Photofrin ® II                                                                      21.3       5          11.9 ± 6.9                                 composition                                                                   BPD-MA    9.2        4          19.0 ± 13.0                                BPD-MB    10.6       3          18.2 ± 11.0                                BPD-DA    10.7       4          18.7 ± 9.9                                 BPD-DB    10.6       3          25.4 ± 16.4                                ______________________________________                                    

Similar studies, except using a light dose of 378 To/cm³, resulted inthe outcome shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                  Number of               Number of                                   Photosensitizer                                                                         Animals    Days Tumor-free                                                                            Cures                                       ______________________________________                                        None      11         0.1          2                                           Photofrin ® II                                                                      10         9.5          4                                           BPD-MA    10         13.2         4                                           BPD-MB     9         8.7          6                                           BPD-DA    15         2.5          4                                           BPD-DB    13         13.0         8                                           ______________________________________                                    

The foregoing results are preliminary, and the assay protocols have notyet been optimized.

EXAMPLE 9 Alternate In Vivo Assay

Mice bearing small tumors were injected IV with drug to be tested. Threehours later the animals were sacrificed and their tumors removed. Thetumor cells were teased apart to form a single cell suspension, and thecells were plated at 10⁵ /well and exposed to light at a prescribeddose. The plates were incubated overnight and assayed for viability byMTT assay.

The results of one study are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                                  Dose                                                                Photosensitizer                                                                         (μg/mouse)                                                                             Light Dose (Jo)                                                                           % Kill                                      ______________________________________                                        BPD-MA    33          5.7         22.0                                                  40          3.8         32.5                                                  80          3.8         63.5 ± 2.1                                         80          3.8         53.7 ± 6.2                               BPD-MB    33          5.7         25.2                                        BPD-DA    80          3.8         11.0                                                  80          7.6         26.0                                        ______________________________________                                    

Thus, the BPD forms tested were active in this assay; it appears lightintensity and drug levels are subject to optimization and correlation.

EXAMPLE 10 Comparison of BPD to Photofrin® II Compositions

Mice bearing P815 tumors were shaved and injected with equivalentamounts of photosensitizer, and exposed to 72 Jo/cm² (80 mw/cm² -15min-full spectrum) at various time intervals. Skin biopsies were takenat 24 and 48 hours after light irradiation and visual evaluations weremade blind. The results of these evaluations are shown in FIG. 4. BPD-MAand, to a lesser extent, BPD-MB had major photosensitizing activity,under these conditions; this was only present when light treatment wasgiven 3 hours post drug administration, consistent with thebiodegradability of these compounds.

EXAMPLE 11 Preparation of Compounds of the Invention

The following compounds have been prepared using the above-describedDiels-Alder reaction of MeOOC-C.tbd.C-COOMe with the dimethyl ester ofprotoporphyrin IX, followed by rearrangement to the forms shown asformulas 3 and 4 of FIG. 1 and by subsequent treatment to hydrolyze ormodify the propionic ester on rings C and D and/or to modify theunreacted vinyl group on the A or B ring remaining after the Diels-Alderreaction with the B or A ring, as the case may be. The products arecompounds of the following formulas, wherein R^(3") is OR* or NR*wherein R* is alkyl, alkylene, or H (or an organic or unorganic cation):##STR12## wherein R¹ and R² are, in all cases, COOMe.

The compounds prepared are as follows:

    __________________________________________________________________________    R.sup.3" (C)                                                                            R.sup.3" (D)                                                                        R.sup.4                                                       __________________________________________________________________________    A-Ring                                                                          OMe     OMe   CHCH.sub.2                                                      OH      OMe   CHCH.sub.2 (BPD-MA)                                             OMe     OH    CHCH.sub.2 (BPD-MA)                                             OH      OH    CHCH.sub.2 (BPD-DA)                                             OMe     OMe   CH(NH.sub.2)Me                                                  OMe     OMe                                                                                  ##STR13##                                                      OH      OH                                                                                   ##STR14##                                                    B-Ring                                                                          OMe     OMe   CHCH.sub.2                                                      OH      OMe   CHCH.sub.2                                                      OMe     OH    CHCH.sub.2                                                      OH      OH    CHCH.sub.2                                                      OMe     OMe   CH(NH.sub.2)Me                                                  OH      OH    CH(NH.sub.2)Me                                                  OMe     OMe   CH(NH(CH.sub.2).sub.6 NH.sub.2)CH.sub.3                         OH      OH    CH(NH(CH.sub.2).sub.6 NH.sub.2)CH.sub.3                         OCD.sub.3                                                                             OCD.sub.3                                                                           CH(NH(CH.sub.2).sub.6 NH.sub.2)CH.sub.3                       10.                                                                             OMe     OMe   CH(imidazolyl)CH.sub.3                                          OMe     OMe                                                                                  ##STR15##                                                      OMe     OMe                                                                                  ##STR16##                                                      OMe     OMe   CH(OH)Me                                                        OMe     OMe   CHBrMe                                                          OMe     OMe   CH(OMe)Me                                                       OMe     OMe   CH(pyridinium Br)Me                                             NH(CH.sub.2).sub.6 NH.sub.2                                                           OMe   CHCH.sub.2                                                      R.sup.3" R.sup.3" NH(CH.sub.2).sub.6 NH                                                     CHCH.sub.2                                                      OMe     OMe   CH(SH)CH.sub.3                                                20.                                                                             OMe     OMe   disulfide of above                                              OMe     OMe   CHO                                                             OMe     OMe   CHOHCH.sub.2 OH                                               __________________________________________________________________________

EXAMPLE 12 Preparation of BPD Dimer-Vinyl Linked

To a stirring solution of BPD-DB (wherein R¹ =R² =carbomethoxy and whichis esterified so that both R³ are carbomethoxyethyl) (35 mg, 48 μmol) in5 ml of dichloromethane cooled to dry ice/acetone temperature was addedtrifluoromethane sulfonic acid (34 μl, 380 μmol). An oil separated outupon the addition of the acid. The reaction was brought up to 0° C. Then5 ml of 5% sodium bicarbonate was added to the reaction to neutralizethe acid. The product distributed into the organic layer which waswashed three times with water. The solvent was removed and the productwas dried via azeotrope with acetonitrile.

Preparative thin layer chromatography on silica gel eluting with 10%ethylacetate/dichloromethane gave a single fraction (28 mg, 80% yield).Parent ion in mass spectrum was 1464. The complex proton NMR due to thenumber of isomeric compounds had the characteristic single vinylhydrogen associated with a C-linkage at about 8.1 ppm.

EXAMPLE 13

Elimination of Viral Contaminants From Blood

Whole blood was obtained from the Canadian Red Cross as units of donorblood. VSV, which was cultured in VHK and VERO cell lines to obtain astock viral concentration of 4×10⁸ plaque-forming units (pfu) /ml, wasadded the blood at a concentration of 10⁷ pfu/ml. The virus-infectedblood was tested using the neutral red dye uptake assay described byFinter, N. B., J Clin Virol (1969) 5:419-425, to establish that, over a6-hour period, no significant nonspecific elimination of active virusoccurred.

VSV-spiked blood was dispensed in 1 ml aliquots into 6-well plates and,under reduced light conditions, varying concentrations of either BPD-MAor BPD-MB were added. The photosensitizers were maintained as stocksolutions of 8 mg/ml in dimethyl sulfoxide (DMSO) at -20° C. They werethawed and diluted immediately before use into phosphate-buffered saline(PBS) before addition to the blood. BPD-MB required a 20-minutesonication in ultrasonic bath to achieve solubility.

The samples were incubated for various time periods in the dark beforeexposure to light, which was provided in a light box containing 16 100 Wtungsten light bulbs (GE 400-1200 nm). The light passed through a 4-cmthick chamber filled with circulating cooled water covered by a mattedglass plate to disperse the light and to give a light intensity of 8mW/cm² as measured by a Gentec Model TPM Radiometer. The 6-well platescontaining the blood samples were placed on top of the matted glassplate and exposed for varying lengths of time.

After treatment, the virus concentration in the blood samples wasdetermined by the neutral red dye uptake assay (Finter (supra)).Briefly, 96-well flatbottom plates were inoculated with VERO cells andgrown to near confluency. Starting at 1:50, fivefold dilutions of thetreated blood samples were added in quadruplicate to the VEROmonolayers. After 48 hours of incubation at 37° C. in 5% CO₂, the plateswere washed twice with PBS and neutral red dye was added and incubatedfor 45 minutes. Excess dye was washed off before addition of lysingbuffer. The color intensity of the dye taken up and retained by viablecells was then measured as absorbance units at 492 nm.

The results of the neutral red dye assay were verified and correlatedwith viral destruction by a standard plaque assay in which dilutions oftreated blood were added (0.1 ml) in duplicate to VERO monolayers in6-well plates. After 1 hour of incubation at 37° C. plus 5% CO₂, themonolayers were washed twice with PBS and overlaid with 0.8% agar inDMEM and plaques were counted after 24 hours.

Parameters for treatment were determined using the foregoing protocol.BPD-MA was added at 1, 2 and 4 μg/ml to 1 and 2 ml of VSV-spiked bloodsamples and the samples were incubated in the dark for 2 hours at roomtemperature before exposure to 57.6 J/cm² of light. In all samples, theaddition of 2 and 4 μg/ml BPD-MA resulted in complete virusinactivation, but at 1 mg/ml, a 4 log virus kill was seen in the 1 mlsample, as compared to only a 2 log virus kill in the 2 ml sample. Theresults show that the blood sample must be sufficiently exposed to thelight source to effect the photosensitization. Consistent with this,when 1 ml samples were spiked with 1, 2 and 4 μg/ml BPD-MA for 2 hoursbefore light exposure for various times, the resulting data showed aninverse relationship between total light energy and the drug doserequired to inactivate the VSV. Furthermore, VSV inactivation by BPD-MAat 1 μg/ml was dramatically improved by increasing light intensity from28.8 to 57.6 J/cm². Exposure to 57.6 J/cm² resulted in 5 logs of kill,as opposed to 2 logs of kill at the lower intensity.

The effect of incubation time prior to light exposure was also testedusing 1 μg/ml BPD-MA at room temperature and incubating at 0.5, 1, 2 and3 hours before exposure to light of 57.6 J/cm². Increase of the drugincubation time from 0.5 hour to 1 hour resulted in improved VSVinactivation, but further increases to 2-3 hours had little effect.Also, little difference was found when BPD-MA was incubated before lightexposure at either room temperature, 37° C. or 4° C.

Finally, the assay was run under standard conditions using 1 ml samplesof VSV-spiked blood treated for 2 hours with various concentrations ofBPD-MA, BPD-MB or commercially available Photofrin® composition prior tolight exposure at 57.6 J/cm². Under these conditions, 2 μg/ml BPD-MAresulted in a 7 log viral kill, 2 μg/ml BPD-MB resulted in 4 logs viralkill, and Photofrin® photosensitizer at even 40 mg/ml did not appear tokill the virus.

Distribution studies showed that approximately 20% of the VSV added tothe samples were associated with the red blood cells, and thatcell-associated VSV particles were inactivated to the same degree asfree virus.

Under the conditions of the assay, the photosensitizer resulted inlittle, if any, lysis of the contained red blood cells.

EXAMPLE 14 Accumulation of BPD in Atherosclerotic Plaques

A. Pig Study

A formulation of BPD-MA was prepared by dissolving 8 mg BPD-MA in 1 mlof DMSO and diluting the resultant 9:1 with normal saline (NS). Theresulting solution was administered intravenously at a dosage of 2 mg/kgto two of three farm swine which previously underwent iliofemoral arterydenudation and had been maintained on a high cholesterol fat diet.

Three hours after the administration of the drug, the animals wereeuthanized and various tissues harvested, including plaque vessel,aorta, heart, kidney, liver, lung, muscle and skin. The tissues wereassayed fluorometrically, including using fluorescence microscopy. Theresults showed that of the two pigs administered BPD-MA, the pig whichhad a high development of plaque disease showed higher accumulation ofthe BPD-MA; highest levels were in the lungs, but it was possible todistinguish between plaqued arteries and unplaqued arteries.

B. In Vitro Human Tissue Study

Human atherosclerotic iliofemoral arteries and aorta were harvested andwrapped in saline-soaked gauze and then incubated for 2 hours at roomtemperature in MEM-Eagle's Medium and 10% fetal calf serum mixed withBPD-MA formulated at 30 μg/ml. The BPD was prepared by mixing 1 ml ofBPD-MA stock solution with 8.1 ml MEM-Eagle's Medium and 0.9 ml fetalcalf serum.

The sample was rinsed three times in cold normal saline, and thesections were examined by fluorescence microscopy to evaluate the uptakeof BPD. The administered dose was finally diluted to provide solutionscontaining 0.5-200 μg/ml, and incubation time was varied. The resultsshowed that incubations performed with 10 μg/ml of BPD-MA or less didnot provide sufficient fluorescence to detect the presence of plaquesusing fluorescence microscopy; however, at 100 μg/ml and above,fluorescence was clearly seen. When approximately 25 μg/ml were used inthe incubation, BPD-MA showed most intensely in the plaqued areas, whileno fluorescence was seen in the underlying arterial wall.

C. Rabbit Studies

In vivo studies were conducted in rabbits which did and did not containatherosclerotic aortas. The rabbits were injected with BPD-MA at variousdosages and time intervals prior to sacrifice. The rabbits wereeuthanized by injection and the entire descending aorta was removed,sectioned, and examined by fluorescence microscopy. No fluorescence wasseen in tissue samples from rabbits receiving 30 μg/kg BPD-MA dosages;however, at 100 μg/kg, whereas no fluorescence was shown in the normalaorta samples, in the two atherosclerotic samples, fluorescence was seenin the plaque but not the arterial wall.

EXAMPLE 15 Detection of Tumors Using BPD-MA

BPD-MA was dissolved in DMSO and diluted with phosphate-buffered salineto a neutral pH and intravenously injected at a dose of 3.5 mg/kg bodyweight to white inbred male Wistar/Furth rats weighing about 240 g whichhad ten days previously been subcutaneously inoculated in both hind legswith syngeneic tumor cells prepared from a colon adenocarcinoma. Therats were sacrificed three hours later and the tumors localized throughthe mediation of the drug.

Fluorescence measurements were made using a fiber optic fluorosensor, asdescribed in Andersson Engels, S., et al., Lasers in Med Sci (1989)4:241. A pulsed nitrogen laser (Laser Science Model VSL-DCM-3) was usedas an excitation source. The laser emits light at 337 nm and every pulseis about 3 ns long at 10 Hertz with a pulse energy of about 175 μJ. Thelight is transmitted through an optical fiber; the same fiber collectsthe fluorescent light which is pulsed through a polychromator with awavelength resolution of 25 nm. A detector is placed in the focal planeof the polychromator. The detector is an EG & G Parc GatableImage-Intensified 1024-Element Diode Array Detector, Model 1421. Thefluorescence signal is detected in the 300-800 nm wavelength range.

The fluorescence spectrum of the emitted light corresponds to that ofBPD-MA. When scanned across a tumor area with surrounding nondiseasedtissue, the BPD-MA-related signal increases over the malignant tumor. Ingeneral, the exterior portion of the tumor, 3 hours after intravenousinjection, has a higher fluorescence than the interior part of the tumorand about 2.5 times higher intensity than the surrounding muscle. Liveralso shows a high fluorescence intensity, while kidney and spleenexhibit low values; the urinary bladder is about the same intensity asthe kidney, and the urine has higher fluorescence. The stomach and smallintestine have higher intensity than the more distal part of thegastrointestinal system, and the feces show very low intensity. Theorgans in the respiratory/circulatory system and skin also have a lowuptake.

The performance of BPD-MA in the detection assay is comparable to thatof the known porphyrin-detecting drugs. Specifically, the ratio of thetumor exterior to muscle for BPD-MA is approximately the same as thatfor HP; however, DHE shows a tumor-to-muscle ratio of 6:1,polyhematoporphyrin ester shows 5.5:1, and tetrasulfonated thalocyanineshows a ratio of about 3:1.

EXAMPLE 16 Effect of Systemic Administration on Papilloma-Caused Warts

Rabbits displaying warts caused by Papilloma virus were providedaccording to the model described by Shope et al., J. Exp. Med. (1933)58:607-624. Five rabbits were administered Photofrin® II porfimer sodiumintravenously at 10 mg/kg total dose over a ≦5 minute period beginning24 hours before irradiation treatment. Six rabbits were administeredBPD-MA intravenously at 2 mg/kg total dose over a ≦5 minute periodbeginning 2.5-3.5 hours before irradiation treatment. Warts wereirradiated individually with no light, 150 J/cm² or 250 J/cm² using 630nm for porfimer sodium and 690 nm for BPD-MA. Controls with no lightirradiation did not show regression.

For 10 warts irradiated at 150 J/cm² in porfimer Na-treated animals, 8completely regressed and another showed >50% regression; for 10 wartsirradiated at 250 J/cm², 8 completely regressed and 2 showed >50%regression.

For 12 warts irradiated at 150 J/cm² in BPD-MA-treated animals, 7completely regressed and 5 showed >50% regression; for 12 wartsirradiated at 250 J/cm², all 12 showed complete cure.

EXAMPLE 17 Topical Treatment of Papilloma-Caused Warts

The efficacy of porfimer Na and BPD-MA applied topically was studied inthe rabbit papilloma virus model of Example 16.

The topical formulations employed were as follows:

1. Photofrin/OAP: 24 mg porfimer sodium per ml vehicle (oleic acid, 23%;ethanol, 46%: propylene glycol, 23%; laureth-9, 8%; and polyvinylpyrrolidone K-90, 20 mg/ml).

2. Photofrin/OB: 39 mg porfimer sodium per ml vehicle (oleyl alcohol,25%; ethanol, 50%; propylene glycol, 25%; benzalkonium chloride, 5mg/ml; polyvinyl pyrrolidone, 20 mg/ml).

3. BPD/Pharmasolve: 10 mg BPD-MA per ml Pharmasolve.

4. BPD/DMSO: 10 mg BPD-MA per ml DMSO.

5. BPD/OA: 10 mg BPD-MA per ml vehicle (a proprietary formulationcontaining oleic acid in an alcohol base, developed by AmericanCyanamid).

6. BPD/OL: 10 mg BPD-MA per ml vehicle (a proprietary formulationcontaining oleyl alcohol in an alcohol base, also developed by AmericanCyanamid).

Each wart was topically treated 3 hours before irradiation with either150 J/cm² or 250 J/cm² at 630 nm for porfimer Na formulations and 690 nmfor BPD formulations.

Photofrin/OAP showed little effect on the warts at either energy;photofrin/OB gave complete regression for 1 of 9 warts at 150 J/cm² and2 of 8 warts at 250 J/cm² ; the remaining warts showed >50% regressionin each case. The results for BPD formulations are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                                  Regression                                                                    100%     >50%       <50%                                            Formulation 150J   250J    150J 200J  150J 250J                               ______________________________________                                        BPD/Pharmasolve                                                                           2/4    2/4     0/4  0/4   2/4  2/4                                BPD/DMSO    0/2    0/2     1/2  0/2   1/2  2/2                                BPD/OA      5/5    6/6     0/5  0/6   0/5  0/6                                BPD/OL      5/7    4/8     2/7  4/8   0/7  0/8                                ______________________________________                                    

Thus, BPD, when properly formulated, is effective in causing regressionapplied topically to warts.

EXAMPLE 18 In Vitro Skin Penetration Studies

Franz in vitro percutaneous diffusion chambers were used to conductstudies of BPD formulations with oleic acid and oleyl alcohol vehicles(¹⁴ C-BPD having a specific activity of 6 μCi was used). Excised splitthickness (200 μ) human cadaver skin was mounted on the chamber (surfacearea 3 cm²). The lower reservoir contained 4% bovine serum albumen inisotonic buffered saline, pH 7.0.

The chambers with the skin were equilibrated to 37° C. prior toapplication of the drug formulations. Formulations (0.25 containing 1.67μCi) were applied to the epidermal surface of the skin. The chamber wascovered (occluded) to prevent evaporation, and the experiment wasconducted in the dark.

Six diffusion cells were run for each time point (1.5 hr, 3 hr, 24 hr)for each formulation. Samples were removed from the dermal chamber at1.5 hr, 3 hr and 24 hr. Cumulative penetration was assayed by liquidscintillation.

At the end of the respective time point, the surface of the skin waswashed to remove surface drug. A 4 mm full thickness skin punch biopsywas taken from each specimen and processed for frozen section tissuefluorescence. This necessitates termination of flux measurements in thatchamber. The stratum corneum was removed by 25 repetitive strippingswith cellophane tape and counted in groups of five. The epidermis washeat-separated from the dermis by heating to 60° C. for 1 minute. Thesetissues were then digested and the ¹⁴ C-BPD content of each of the skincompartments and reservoir was determined by liquid scintillationcounting. Results are set forth in Tables 10 and 11. (In the tables,"BPD-OA" and "BPD-OL" represent the BPD-oleic acid and BPD-oleyl alcoholformulations of the preceding example, "CH-014" represents ¹⁴ C-labeledBPD-MA, and "phospholipid" represents a BPD-MA liposomal formulation.)

                                      TABLE 10                                    __________________________________________________________________________    PERCENT RECOVERY                                                              __________________________________________________________________________            Stratum Corneum   Epidermis                                           TIME (HRS):                                                                           1.5   3     24    1.5  3    24                                        __________________________________________________________________________    BPD-OA  15.5 ± 11.4                                                                      16.6 ± 1.8                                                                       11.6 ± 7.5                                                                       .12 ± .04                                                                       .10 ± .03                                                                       .08 ± .09                              BPD-OL  4.9 ± 1.6                                                                        16.1 ± 10.2                                                                      11.1 ± 5.0                                                                       .08 ± .03                                                                       .12 ± .04                                                                       .05 ± .02                              CH-014                    .13 ± .13                                                                       .06 ± .02                                                                       .06 ± .03                              Phospholipid              .21 ± .13                                                                       .28 ± .13                                                                       .24 ± .19                              __________________________________________________________________________            Dermis            Reservoir                                           TIME (HRS):                                                                           1.5   3     24    1.5  3    24                                        __________________________________________________________________________    BPD-OA  .34 ± .10                                                                        .37 ± .07                                                                        .36 ± .17                                                                        0    .01 ± .01                                                                       .08 ± .03                              BPD-OL  .25 ± .10                                                                        .30 ± .12                                                                        .25 ± .08                                                                        0    0    .11 ± .06                              CH-014  .19 ± .06                                                                        .32 ± .11                                                                        .40 ± .28                                                                        0    0    .014 ± .018                            Phospholipid                                                                          .48 ± .14                                                                        .62 ± .36                                                                        .30 ± .12                                                                        0    0    .014 ± .002                            __________________________________________________________________________

                  TABLE 11                                                        ______________________________________                                        TISSUE FLUORESCENCE                                                                   Strateum Corneum                                                                          Epidermis  Dermis                                         TIME (HRS):                                                                             1.5    3       24   1.5 3    24  1.5 3   24                         ______________________________________                                        BPD-OA    2.7    2.7     3    0.5 0.7  1.2 0.7 0.7 1.7                        BPD-OL    3      2.8     3    1.5 1.8  2.5 1.2 1.5 2.3                        CH-014    2.5    3       3    1.2 1.8  1.4 1   1   1                          Phospholipid                                                                            3      3       3    1.2 1.8  2.2 1.2 1.3 1.6                        ______________________________________                                    

EXAMPLE 19 LED Activation of BPD

Light Emitting Diodes (LEDs) which emit light around 690 nm were used toactivate BPD for viral inactivation in blood (red cell concentrates).Treatment of red cell concentrates was as follows.

Red cell concentrates (Adsol) were spiked with 6 logs of VesicularStomatitis Virus (VSV) per ml. After incubation with virus, samples wereincubated for 60 minutes with BPD at 1 μg/ml or 0.5 μg/ml. The sampleswere washed twice and placed in red cell storage solution, then exposedto red LED light from each of two sides for 5, 10 or 15 minutes(approximately 7, 14 or 22 Joules/cm²). The samples were assayed forremaining infectious virus by a standard TCID 50 (tissue cultureinfectious dose) assay. Results showed no recoverable virus in any ofthe samples that had been treated with both BPD and light.

The advantages of using these LEDs compared to other non-laser lightsources are, first of all, increased power output (20-24 mW/cm²)compared to red fluorescent lights (approximately 2 mW/cm²). The LEDlight used in this example was focused around the optimal wavelength(690 nm) for activation of BPD in blood. Secondly, markedly, the LEDsused in the aforementioned method gave rise to markedly decreasedtreatment times (5-10 minutes or less, depending on dose of BPD).Previous treatment times to achieve 100% viral inactivation in thissystem are on the order of 1-2 hours.

FIGS. 5, 6 and 7 demonstrate results from a variety of experiments toevaluate effects of BPD concentration and light exposure times (lightdose) on viral kill in plasma (FIG. 5) or red cell concentrations (FIGS.5, 6 and 7). A slightly different treatment method was utilized forthese studies than that outlined above. In these studies, BPD doses ofgreater than or equal to 1 μg/ml and light doses of betweenapproximately 6 and 14 Joules/cm² resulted in 100% viral kill.

FIGS. 8, 9 and 10 demonstrate results from recent experiments utilizingtwice the light intensity (48 mW/cm²) of that utilized in previousstudies. These graphs demonstrate that 100% viral kill can be achievedby the use of 0.5 μg BPD/ml and 5 minutes light exposure (approximately14 Joules/cm²) or 0.25 μg BPD/ml and 10 minutes light exposure(approximately 29 Joules/cm²).

We claim:
 1. A method for treating skin diseases in an animal whichcomprises topically treating an animal in need of such treatment with aneffective amount of a hydro-monobenzoporphyrin (Gp) having a lightabsorption maximum between 670-780 nm;wherein the Gp is selected fromthe group consisting of ##STR17## and mixtures thereof and the metalatedand labeled forms thereof, wherein each R¹ and R² independently selectedfrom the group consisting of carbalkoxy (2-6C), alkyl (1-6C) sulfonyl,aryl (6-10C) sulfonyl, aryl (6-10C); cyano; and --CONR⁵ CO-- wherein R⁵is aryl (6-10C) or alkyl (1-6C); each R³ is independently carboxyalkyl(2-6C) or a salt, amide, ester or acylhydrazone thereof, or is alkyl(1-6C); and R⁴ is CHCH₂, CH₂ OR^(4'), --CHO, --COOR^(4'),CH(OR^(4'))CH₃, CH(OR^(4'))CH₂ OR^(4'), --CH(SR^(4'))CH₃, --CH(NR^(4')₂)CH₃, --CH(CN)CH₃, --CH(COOR^(4'))CH₃, --CH(halo)CH₃, or --CH(halo)CH₂(halo), wherein R^(4') is H or alkyl (1-6C) optionally substituted witha hydrophilic substituent; or wherein R⁴ is an organic group of <12Cresulting from direct or indirect derivatization of vinyl; or wherein R⁴consists of 1-3 tetrapyrrole-type nuclei of the formula --L--P wherein--L-- is selected from the group consisting of ##STR18## and P isselected from the group consisting of Gp which is of the formula 1-6 butlacking R⁴ and conjugated through the position shown as occupied by R⁴to L, and a porphyrin of the formula: ##STR19## wherein each R isindependently H or lower alkyl (1-4C); wherein two of the bonds shown asunoccupied on adjacent rings are joined to R³ and one of the remainingbonds shown as unoccupied is joined to R⁴ and the other to L; with theproviso that if R⁴ is CHCH₂, both R³ cannot be carbalkoxyethyl; andirradiating the animal with light absorbed by said Gp.